CDH6 Antibody

What is CDH6 and what is its expression pattern in normal versus malignant tissues?

CDH6, also known as K-cadherin, is a type II classic cadherin molecule that functions in cell-cell adhesion. It plays an important role in embryonic kidney development but demonstrates very limited expression in normal adult tissues . In contrast, CDH6 is overexpressed in several human malignancies, primarily ovarian and renal cancers, but also (to a lesser degree) in gastric, thyroid, and cholangiocarcinoma . This differential expression pattern makes CDH6 an attractive target for cancer therapeutics, particularly antibody-drug conjugates.

The limited expression in normal tissues combined with high expression in tumor tissues creates a potentially favorable therapeutic window. Additionally, CDH6 undergoes rapid internalization upon antibody binding, further enhancing its suitability as an ADC target .

What experimental approaches are used to validate CDH6 as a therapeutic target?

To validate CDH6 as a therapeutic target, researchers employ multiple complementary approaches:

• Expression analysis: Using techniques such as RNA sequencing, western blotting, and immunohistochemistry to confirm differential expression between normal and cancer tissues.

• Genetic manipulation: CRISPR/Cas9-mediated knockout of CDH6 in cancer cell lines to assess its biological function. As described in the literature, researchers have designed targeted sequences for exon 2 and intron 3 based on the genomic sequence of human CDH6 (Gene ID: 1004) .

• Antibody development and characterization: Generation of anti-CDH6 antibodies through techniques like in vivo electroporation followed by hybridoma fusion, with screening for selective binding to CDH6 .

• Xenograft models: Both cell line-derived xenografts (CDX) and patient-derived xenografts (PDX) are used to evaluate the efficacy of CDH6-targeting therapies in vivo .

What approaches are used to generate CDH6-specific antibodies?

The generation of CDH6-specific antibodies involves several sophisticated methodologies:

• Immunization strategies: Rats can be immunized with human CDH6-expressing vectors administered intramuscularly, followed by in vivo electroporation using systems like ECM830 Square Wave Electroporation System .

• Hybridoma technology: After immunization, lymph node cells from animals with high antibody titers are harvested and fused with myeloma cells (e.g., SP2/0-ag14) using electrofusion techniques like Hybrimune Hybridoma Production Systems .

• Screening: Hybridoma supernatants are screened for selective reactivity with human CDH6 using Cell ELISA with CDH6-expressing cells. Positive clones are further tested for binding to both human and non-human primate (e.g., cynomolgus monkey) CDH6 to assess cross-reactivity .

• Humanization: Rodent antibodies can be humanized by sequencing the cDNA of promising clones and redesigning them as humanized IgG antibodies, as demonstrated with the G019 H1L2 antibody .

How are CDH6 antibodies validated for specificity and affinity?

Validation of CDH6 antibodies requires rigorous testing through multiple complementary approaches:

• Flow cytometry: To confirm binding to CDH6-expressing cells and absence of binding to CDH6-knockout cells .

• Western blotting: Using recombinant CDH6 protein as a positive control and CDH6-knockout cells as negative controls .

• Immunoprecipitation: To verify that the antibody can pull down CDH6 and its associated proteins like α-catenin and β-catenin .

• Binding assays: ELISA-based methods to determine binding kinetics and affinity constants for the antibody-antigen interaction .

• Cross-reactivity testing: Evaluating binding to CDH6 from different species (e.g., human versus cynomolgus monkey) to assess cross-reactivity for translational research .

What are the key considerations in designing CDH6-targeting antibody-drug conjugates?

Designing effective CDH6-targeting ADCs requires optimization of multiple components:

• Antibody selection: The antibody must demonstrate high specificity, appropriate affinity, and efficient internalization upon CDH6 binding .

• Linker chemistry: The choice between cleavable and non-cleavable linkers significantly impacts the ADC's mechanism of action and efficacy. For example, protease-cleavable linkers are commonly used in CDH6 ADCs, as seen with CUSP06 .

• Payload selection: Different cytotoxic payloads confer distinct properties:

  • DXd (deruxtecan): A potent DNA topoisomerase I inhibitor used in R-DXd

  • Exatecan: Used in CUSP06/AMT-707, shown to demonstrate improved bystander effects compared to DXd-based ADCs

  • DM4: Used in HKT288, selected after optimization studies

• Drug-to-antibody ratio (DAR): Optimizing the number of drug molecules conjugated to each antibody, with CUSP06 having a DAR of 8 .

How do preclinical models inform the development of CDH6-targeting ADCs?

Several preclinical model systems are instrumental in developing CDH6-targeting ADCs:

• Cell line panels: Testing ADCs against multiple CDH6-expressing cancer cell lines to determine in vitro potency and selectivity .

• Cell line-derived xenografts (CDX): Evaluating efficacy in vivo using established cancer cell lines like PA-1, OVCAR3, and 786-O .

• Patient-derived xenografts (PDX): More clinically relevant models that maintain tumor heterogeneity. For example, HKT288 development incorporated a population-based PDX clinical trial (PCT) with 30 unselected models to better predict patient response heterogeneity .

• Toxicology studies: Assessing safety profile and on-target/off-target effects in appropriate animal models. For instance, CUSP06 showed an expected toxicity profile consistent with its exatecan payload in pilot toxicology studies .

How should researchers address species differences in CDH6 expression when designing preclinical studies?

The significant species differences in CDH6 expression present important challenges for preclinical research:

• Expression pattern discrepancies: CDH6 is present on human platelets (~4500 copies per platelet) but absent on mouse platelets, as confirmed by western blotting and flow cytometry . This creates challenges for translating mouse model results to humans.

• Experimental approaches to address these differences:

  • Use multiple mouse strains (e.g., C57BL/6J, FVB, 129x1/SVJ) to confirm expression patterns

  • Incorporate RNA sequencing data from both human and mouse tissues

  • Consider humanized mouse models expressing human CDH6

  • Include non-human primate studies where feasible, as cynomolgus monkey CDH6 may better represent human CDH6 biology

• Experimental controls: Always include both CDH6 knockout models and species-appropriate positive controls when evaluating antibody specificity .

What experimental approaches best characterize the bystander effect of CDH6-targeting ADCs?

The bystander effect—where ADCs can kill neighboring tumor cells regardless of target expression—is particularly important for heterogeneously expressed targets like CDH6:

How can population-based PDX clinical trials enhance the development of CDH6-targeting therapies?

Population-based PDX clinical trials (PCTs) represent an important advancement in preclinical evaluation:

• Representative heterogeneity: By incorporating multiple PDX models (e.g., 30 unselected models as used for HKT288), PCTs capture the heterogeneity of response across a patient population .

• Experimental design considerations:

  • Include models with varying levels of CDH6 expression

  • Define clear endpoints (e.g., tumor regression, stable disease)

  • Standardize dosing and administration schedules

  • Include appropriate control groups

• Biomarker identification: PCTs facilitate retrospective biomarker analysis to identify predictors of response, which can guide patient selection criteria for clinical trials .

• Translational value: The response rate in a well-designed PCT (e.g., 40% of models for HKT288) may provide a preliminary estimate of clinical activity .

What are the optimal methods for detecting and quantifying CDH6 expression in research and clinical samples?

Accurate detection and quantification of CDH6 is critical for research and potential patient selection:

• Western blotting optimization:

  • Use purified recombinant CDH6 to generate standard curves for quantification

  • Include positive controls (e.g., CDH6-transfected cells) and negative controls (CDH6 knockout cells)

  • Can detect as few as 300 copies per cell with optimized protocols

• Flow cytometry:

  • APC-conjugated anti-CDH6 antibodies can be used to assess surface expression

  • Compare signal between wild-type and CDH6 knockout cells to confirm specificity

• Immunohistochemistry:

  • Optimize antigen retrieval methods for formalin-fixed, paraffin-embedded tissues

  • Use scoring systems that account for both intensity and percentage of positive cells

• RNA analysis:

  • RNA sequencing or qRT-PCR for mRNA expression

  • Consider both absolute expression and relative expression compared to normal tissues

What strategies can address potential on-target, off-tumor toxicity of CDH6-targeting therapies?

Managing on-target, off-tumor toxicity requires careful consideration:

• Comprehensive tissue expression profiling:

  • Evaluate CDH6 expression across normal human tissues using techniques like RNA sequencing and immunohistochemistry

  • Special attention to platelets, which express CDH6 in humans but not in mice

• Linker optimization:

  • Selecting linkers with appropriate stability to minimize premature drug release in circulation

  • The preclinical development of HKT288 specifically focused on linker optimization to improve therapeutic index

• Dosing strategies:

  • Consider intermittent dosing schedules to allow recovery of normal CDH6-expressing tissues

  • Establish minimum effective dose through careful dose-response studies

• Alternative formats:

  • Explore bispecific antibodies requiring dual-antigen binding for activation

  • Consider masked antibody approaches that become activated in the tumor microenvironment

How might CDH6 antibodies be used beyond direct targeting of cancer cells?

CDH6 antibodies have potential applications beyond direct cancer cell targeting:

• Vascular biology: Given the role of CDH6 in thrombosis , CDH6 antibodies may have applications in studying and potentially modulating vascular function.

• Imaging applications: CDH6 antibodies conjugated to imaging agents could facilitate tumor detection and monitoring of CDH6-expressing cancers.

• Combinatorial approaches: CDH6 antibodies could be combined with immune checkpoint inhibitors or other targeted therapies. Studies should evaluate potential synergistic effects through careful in vitro and in vivo experimental designs.

• CAR-T cell therapy: CDH6-targeting single-chain variable fragments could be incorporated into chimeric antigen receptor constructs for adoptive T cell therapy of CDH6-expressing tumors.

What are the implications of CDH6's association with catenins for cancer biology research?

The association of CDH6 with α-catenin and β-catenin opens several research directions:

• Signaling pathway analysis:

  • Investigate how CDH6-catenin complexes affect Wnt/β-catenin signaling

  • Examine effects on cell adhesion, migration, and epithelial-mesenchymal transition

• Experimental approaches:

  • Co-immunoprecipitation to identify additional proteins in the CDH6-catenin complex

  • Proximity ligation assays to visualize protein-protein interactions in situ

  • Live cell imaging to track dynamics of CDH6-catenin interactions

• Functional studies:

  • Compare effects of CDH6 antibodies that do or do not disrupt catenin associations

  • Evaluate downstream signaling consequences of CDH6 targeting in the context of catenin pathway activation status